Peroxynitrite is a potent NO-derived oxidant and important mediator of inflammation, ischemia- reperfusion injury and shock. Peroxynitrite can cause both apoptotic and at higher concentrations necrotic cell death. This latter process involves the activation of the nuclear enzyme poly (ADP-ribose) synthetase/polymerase (PARS or PARP) that becomes activated in response to DNA damage and contributes to necrosis via depletion of its substrate NAD and consequently of ATP. In the previous funding cycle, we have identified several key aspects of peroxynitrite-mediated cell death, and demonstrated the central role of PARS in this process. Among other mechanisms, we have obtained data demonstrating the role of disturbances in cellular calcium homeostasis during peroxynitrite-induced cytotoxicity. In the current continuation project, we will investigate, in further detail, some of the key aspects of peroxynitrite cytotoxicity. Specific aims: #1. To delineate the mechanisms by which calcium signaling plays a role in the peroxynitrite- induced cytotoxicity. Our preliminary data indicate that peroxynitrite triggers a rapid) rise of Cai++ , the inhibition of which (by cell- permeable Cai++ chelators) reduces peroxynitrite-elicited PARS activation. We propose studies, which will identify the sources of Cai++ mobilization during peroxynitrite toxicity. #2. To establish the role of intracellular calcium signals in the regulation of peroxynitrite-induced DNA single strand breakage and PARS activation. We will investigate whether peroxynitrite directly or through the calcium dependent mitochondrial formation of secondary radicals causes DNA single strand breakage and how calcium signals are involved in PARS activation. #3. To establish the role of protein kinase C (PKC) in the regulation of peroxynitrite-induced cytotoxicity with special regard to PARS activation. Our preliminary data indicate that in thymocytes, peroxynitrite-induced PARS activation is inhibited by PKC activators. We wish to investigate whether peroxynitrite activates PKC and what PKC isozymes are involved in the regulation of of peroxynitrite induced cell death and of PARS activity. #.4 To establish the role of Bel family proteins in the regulation of calcium homeostasis during peroxynitrite-induced cell death. The studies proposed here have broad pathophysiological relevance.